Our involvement in the regulation and consequences of ubiquitination began in the course of studies aimed at understanding why double positive (DP) thymocytes are so sensitive to pro-apoptotic stimuli. One clue was that DP apoptosis is blocked by proteasome inhibitors, implicating regulated protein degradation as a sensitizing event. We found that induction of DP apoptosis, regardless of the molecular pathway, resulted in the degradation of XIAP and c-IAP1, proteins of the Inhibitor of APoptosis (IAP) family. Importantly, we identified XIAP and c-IAP1 as ubiquitin protein ligases (E3s), enzyme involved in the addition of Ub to target proteins. This activity was dependent upon a motif called the RING domain. In subsequent studies we made the following findings: - Signaling via Tumor Necrosis Factor (TNF) receptor 2 (TNF-R2), but not TNF-R1, results in ubiquitination and degradation of the signaling intermediate TRAF2 (TRAF2 is required for coupling the TNF-R to JNK activation and, with TRAF5, to NF-kappaB). - TNF-R2 -mediated ubiquitination of TRAF2 is mediated by c-IAP1. - Expression of an "E3-dead" c-IAP1 RING point mutant (a dominant negative) prevented TNF-alpha-induced TRAF2 degradation and inhibited apoptosis, demonstrating that c-IAP1 can actually be pro-apoptotic, probably by causing the degradation of TRAF2 and, perhaps, other anti-apoptotic molecules. - Stimulation via TNF-R2 results in the translocation of a c-IAP1/TRAF2 complex to the perinuclear ER, where it encounters a ubiquitin conjugating enzyme (E2), which cooperates with c-IAP1 to cause TRAF2 ubiquitination. - We were the first to characterize mice deficient in c-IAP1, and found that it has an obligate role in the ubiquitination and degradation of another member of the IAP family, c-IAP2. We have also found that ASK1, an important upstream enzyme in the MAP kinase signaling cascade, is a target for c-IAP1 in B cells stimulated with TNF. As a result, MAP kinase signaling is terminated in a timely fashion, moderating B cell responses. Another area in which we have studied ubiquitination is in signaling for activation of the important transcription factor NF-kappaB. NF-kappaB is sequestered in the cytoplasm in a complex with IkappaB. Almost all NF-kappaB activation pathways converge on IkappaB kinase (IKK), which phosphorylates IkappaB resulting in I-B K48-linked polyubiquitination, IkappaB degradation by proteasomes, and migration of NF-kappaB to the nucleus. IKK has two enzymatically-active subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma or NEMO. NEMO is essential for NF-kappaB activation, and NEMO mutations or deficiency have been identified as the cause of incontinentia pigmenti (IP) and hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). The mechanism by which proximal cytokine receptor signals result in its NEMO-dependent activation remains largely unknown. Among the best-studied of such signaling pathways is that for TNF-alpha. TNF receptor 1 (TNF-R1) occupancy results in receptor trimerization and the serial recruitment of TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), receptor-interacting protein (RIP), TRAF2, and c-IAP1 and c-IAP2. RIP in particular is an essential intermediate for downstream activation of NF--B. Upon stimulation with TNF-alpha, RIP binds to NEMO, which brings with it the other IKK components. The RIP that associates with TNF-R1 undergoes polyubiquitination, initially K63-linked, in lipid rafts; the K63-linked polyUb is subsequently removed by the de-ubiquitinating domain of A20 and K48-linked polyUb chains are added by the zinc finger region of A20, resulting in RIP degradation. We used NEMO in a yeast two-hybrid screen to look for interacting proteins, and identified 2-3 residue multi-ubiquitin as a binding partner. In depth analysis identified a motif in NEMO that bound K63-linked (but not K48-linked) polyUb. Moreover, NEMO specifically bound polyUb-modified RIP in TNF-signaled cells, which was prevented by mutations in the NEMO binding site. These same mutations have been identified as causing HED-ID in humans. This led us to conclude that one major function of NEMO is to act as a sensor of K63-linked polyUb, which explains why polyUb is necessary for signaling for NF-kappaB activation in the TNF pathway. Optineurin is a protein whose mutation is responsible for a subset of adult-onset primary open angle glaucoma. Optineurin contains a motif highly homologous to the Ub-binding motif in NEMO. In fact, we have found the optineurin binds to K63-linked polyUb much better than NEMO, and that it competes with NEMO for ubiquitinated RIP in TNF-stimulated cells. Acquisition of optineurin inhibits NF--B activation, and forced knock-down of optineurin greatly enhances NF--B activation. Given that NF-kappaB greatly enhances excitotoxic neuronal cell death, we have proposed that loss-of-function mutations in optineurin may in fact cause glaucoma due to enhanced retinal neuron cell death. We have also been studying other signaling pathways that result in NF-kappaB activation, in particular IL-1 receptor (IL-1R)/Toll-like receptor and T cell receptor (TCR) signaling. We have found that both types of receptor use a strategy similar to that of TNF. In particular, in each a particular molecule in the signaling complex is polyubiquitinated with K63-linked chains, which results in the recruitment of NEMO and activation of IKK and NF--B. Disruption of this, either by mutating the binding site in NEMO or the ubiquitination sites in the adaptor molecules, greatly diminished NF--B activation. These results establish our finding that NEMO recognizes K63-linked polyUb chains as a general and evolutionarily conserved mechanism for activating NF-kappaB in response to extracellular stimuli. Given the central importance of NF--B in immune and inflammatory responses, this identifies NEMO/polyUb binding as a possible molecular target of intervening in these processes. We have generated mice in which we "knocked-in" an E3-defective c-IAP2 (it contains a point mutation in the RING domain). These animals exhibit an expansion of lymphocytes, in particular B cells, in the secondary lymphoid organs. This seems to be accompanied by an spontaneous upregulation of NF-kappaB activity, and is linked to increased B cell survival in vitro. We are currently testing the possibility that c-IAP2 regulates signaling by B cell survival factors and that in its functional absence there is too much signaling. Given that a subset of human B cell lymphomas, known as MALT lymphomas, have a translocation that renders c-IAP2 enzymatically inactive, this is a potentially very useful pre-clinical model system. We have also generated optineurin knockin mice that lack the first 129 amino acids. We have found that optineurin binds two kinases, Tank-binding kinase 1 and IKKepsilon, involved in the production of Type I interferons, and thus anti-viral responses. Because these kinases bind the amino-terminal half of optineurin, we hope that these mice will provide a useful model for studying the role of optineurin in viral responses.